Hoechst33342 disrupts the interaction of Gfi-1 or Gfi-1 like proteins with the 28-bp AT-rich DNA element (H369W)

<p><b>Copyright information:</b></p><p>Taken from "Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance"</p><p></p><p>Nucleic Acids Research 2007;35(7):2390-2402.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874665.</p><p>© 2007 The Author(s)</p> () H369W harbors a Gfi-1-like DNA-binding site. () Hoechst33342 abrogates the interaction of Gfi-1 or Gfi-1 like proteins with the H369W DNA element. DNA–protein-binding reactions were run on a 4% non-denatured PAGE gel. Combinations of the H369W DNA element probe with nuclear extracts, Hoechst33342, specific DNA competitors (cold H369W or cold Gfi-1) and non-specific competitors (scramble DNA oligonucleotides) as well as two other ligands (DAPI and Distamycin) are indicated. () Gfi-1 antibodies supershift the upper H369W–protein complex. The experiment condition is as in (B). As indicated, in the presence of anti-Gfi-1 antibody, the upper DNA–protein complex was supershifted (see Discussion section for the lower band). () Hoechst33342 strongly induces survivin promoter activity in pLuc-957 possessing the 28-bp DNA element, while it has no significant effect on survivin promoter activity in the pLuc-839 construct without the 28-bp DNA element. The histogram data were derived from three independent experiments. Each bar is the mean ± SD. () and () Hoechst33342 induces Gfi-1 mRNA expression (E), while it has no significant effect on Gfi-1 protein expression (F). Data in (E) represents real-time QPCR from three independent testing. Each bar is the mean ± SD. Data in (F) is western blot analysis. Actin is the internal protein loading control

A low concentration of Hoechst33342 protects cells from death induced by hedamycin

<p><b>Copyright information:</b></p><p>Taken from "Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance"</p><p></p><p>Nucleic Acids Research 2007;35(7):2390-2402.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874665.</p><p>© 2007 The Author(s)</p> () HeLa cells were equally seeded in 24-well plates. Cells grown to 70–80% confluence were treated without (a) and with Hoechst33342 (b), hedamycin (c) or Hoechst33342/hedamycin combination (d) as shown. Note: Hoechst33342 was added 2 h before adding hedamycin. Images were taken 36 h after adding hedamycin. () Trypan blue exclusion assays were used to count the number of alive cells after treatment in (A). Data presented in a histogram are the mean ± SD derived from three independent well countings. () Modulation of survivin promoter activity by hedamycin (Hed) and Hoechst33342 (Hoe) alone and in combination. HeLa cells were transfected with survivin promoter-luciferase construct pLuc-1430. Cells were treated with Hed (0.5 and 1 nM) and Hoe (250 nM) alone or in combination as shown. Luciferase activity was measured 36 h after drug treatment. Each bar in the histogram is the mean ± SD derived from three independent testings. Seq, sequentially (Hed was added to cells after Hoe treatment for 2 h); con, concurrently

Mangiferin Accelerates Glycolysis and Enhances Mitochondrial Bioenergetics

One of the main causes of hyperglycemia is inefficient or impaired glucose utilization by skeletal muscle, which can be exacerbated by chronic high caloric intake. Previously, we identified a natural compound, mangiferin (MGF) that improved glucose utilization in high fat diet (HFD)-induced insulin resistant mice. To further identify the molecular mechanisms of MGF action on glucose metabolism, we conducted targeted metabolomics and transcriptomics studies of glycolyic and mitochondrial bioenergetics pathways in skeletal muscle. These data revealed that MGF increased glycolytic metabolites that were further augmented as glycolysis proceeded from the early to the late steps. Consistent with an MGF-stimulation of glycolytic flux there was a concomitant increase in the expression of enzymes catalyzing glycolysis. MGF also increased important metabolites in the tricarboxylic acid (TCA) cycle, such as α-ketoglutarate and fumarate. Interestingly however, there was a reduction in succinate, a metabolite that also feeds into the electron transport chain to produce energy. MGF increased succinate clearance by enhancing the expression and activity of succinate dehydrogenase, leading to increased ATP production. At the transcriptional level, MGF induced mRNAs of mitochondrial genes and their transcriptional factors. Together, these data suggest that MGF upregulates mitochondrial oxidative capacity that likely drives the acceleration of glycolysis flux

Mapping the survivin promoter region that mediates Hoechst33342's effects on the induction of survivin promoter activity

<p><b>Copyright information:</b></p><p>Taken from "Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance"</p><p></p><p>Nucleic Acids Research 2007;35(7):2390-2402.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874665.</p><p>© 2007 The Author(s)</p> () The 781-bp DNA region between pLuc-1430 and pLuc-649 was identified to mediate the upregulation of survivin promoter activity by Hoechst33342. HeLa cells were transfected with various survivin promoter-luciferase constructs as shown and treated with or without Hoechst33342 (5 μM) 24 h after transfection. Cells were lysed and luciferase activities were determined 24 h after treatment. () Nested deletion of the 781-bp DNA region between pLuc-1430 and pLuc-649 identified a 117-bp DNA region mediating a major effect of Hoechst33342. Transfection, drug treatment and luciferase assay are as in (A). In both (A) and (B), luciferase activities were normalized to Renilla luciferase internal controls as arbitrary units and are shown as a histogram. Each bar is the mean ± SD from the experiment in triplicate

Footprinting identified the alteration of DNA–protein interactions in an AT-rich DNA element in the functionally identified 117-bp fragment after Hoechst33342 treatment

<p><b>Copyright information:</b></p><p>Taken from "Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance"</p><p></p><p>Nucleic Acids Research 2007;35(7):2390-2402.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874665.</p><p>© 2007 The Author(s)</p> () DNA sequences from −779 to −1108 containing the functionally identified 117-bp DNA fragment. The primers used for LM-PCR and the 28-bp AT-rich DNA element in bold are indicated. () Hoechst33342 protected an AT-rich DNA element from DMS-mediated piperidine digestion. HeLa cells were treated with (lane 2) or without (lane 1) 50 nM Hoechst33342 for 16 h and then processed DMS-mediated piperidine digestions and LM-PCR. Positions of the AT-rich DNA element and the protected band in lane 1 from forward (atFP3) and reverse (atFPr3) directed LM-PCR are indicated

Comparison of the cytotoxicity of Hoechst33342 with hedamycin HeLa cells were seeded in 24-well plates and grown to a 50% of confluence

<p><b>Copyright information:</b></p><p>Taken from "Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance"</p><p></p><p>Nucleic Acids Research 2007;35(7):2390-2402.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874665.</p><p>© 2007 The Author(s)</p> Cells were then treated with and without various concentration of Hoechst33342 () or hedamycin () as shown. Cell images were taken under an inverted phase-contrast microscope with a digital camera at 24 and 48 h after treatment. () HeLa cell growth inhibition after Hoechst33342 treatment. Cells were seeded in 96-well plates and grown to ∼40% of confluence. Cells were then treated with and without Hoechst33342, as shown. Cell viability was determined by MTT assay. Absorbance at 570 nm for the control (no Hoechst33342) is set as 1 and results are reported in a histogram (the mean ± SD from 5–10 measurements for each point)